Listeria monocytogenes (Lm) is a foodborne intracellular pathogen. There are ~2500 cases of listeriosis/yr and the mortality rate is 20-30%. Our understanding of how pathogens like Lm adapt their metabolism to grow successfully in a host is still rudimentary. The goal of this study is to define mechanisms by which bacteria replicate in the host cell environment. Only two genes are known to contribute to Lm intracellular growth, hpt and lplA1. LplA1 encodes a lipoate protein ligase (lpl);delta-lplA1 mutants have a 250-fold virulence defect. Lpls provide a critical co-factor, lipoic acid (LA), to the cell in a usable form for aerobic metabolism. Lm has two lpl genes, lplA1 and lplA2;little is known about their functional importance in Lm pathogenesis. Previous work showed that host derived LA (HDLA) is essential for intracellular growth and virulence of Lm. We hypothesize that LplA1 is critical for use of HDLA while LplA2 uses free LA as a substrate. This proposal focuses on elucidating how Lm acquires and uses LA during infection. 1) The roles of LplA1 and LplA2 in growth in vitro and in host cells will be determined. Strains deficient in lplA1, lplA2 or both will be analyzed for extracellular and intracellular growth, virulence, and resistance to oxidative stress. 2) The substrates and targets for LplA1 and LplA2 will be identified. In vitro growth of Lm using different host derived substrates will be examined. In addition, bacterial proteins that we have observed are lipoylated only during intracellular growth will be identified by mass spectrometry. 3) The transporter for HDLA will be identified and characterized. The mechanism of transport of HDLA is unknown. A validated genetic selection will be used to identify Lm mutants that have a phenotype like delta-lplA1. Overall, these studies will reveal essential adaptations that permit Lm to exploit the host cell niche. In addition, the characterization of Lm mutants that are defective in intracellular growth will provide new candidates for live vaccine development and may identify new targets for anti-microbial therapeutics.